At mammalian body temperature, the plague bacillus Yersinia pestis synthesizes lipopolysaccharide (LPS)-lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity. To address the effect of weak TLR4 stimulation on virulence, we modified Y. pestis to produce a potent TLR4-stimulating LPS. Modified Y. pestis was completely avirulent after subcutaneous infection even at high challenge doses. Resistance to disease required TLR4, the adaptor protein MyD88 and coreceptor MD-2 and was considerably enhanced by CD14 and the adaptor Mal. Both innate and adaptive responses were required for sterilizing immunity against the modified strain, and convalescent mice were protected from both subcutaneous and respiratory challenge with wild-type Y. pestis. Despite the presence of other established immune evasion mechanisms, the modified Y. pestis was unable to cause systemic disease, demonstrating that the ability to evade the LPS-induced inflammatory response is critical for Y. pestis virulence. Evading TLR4 activation by lipid A alteration may contribute to the virulence of various Gram-negative bacteria.

Transcribed, low-copy repeat elements are associated with the breakpoint regions of common deletions in Prader-Willi and Angelman syndromes. We report here the identification of the ancestral gene ( HERC2 ) and a family of duplicated, truncated copies that comprise these low-copy repeats. This gene encodes a highly conserved giant protein, HERC2, that is distantly related to p532 (HERC1), a guanine nucleotide exchange factor (GEF) implicated in vesicular trafficking. The mouse genome contains a single Herc2 locus, located in the jdf2 (juvenile development and fertility-2) interval of chromosome 7C. We have identified single nucleotide splice junction mutations in Herc2 in three independent N-ethyl-N-nitrosourea-induced jdf2 mutant alleles, each leading to exon skipping with premature termination of translation and/or deletion of conserved amino acids. Therefore, mutations in Herc2 lead to the neuromuscular secretory vesicle and sperm acrosome defects, other developmental abnormalities and juvenile lethality of jdf2 mice. Combined, these findings suggest that HERC2 is an important gene encoding a GEF involved in protein trafficking and degradation pathways in the cell.

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The juvenile development and fertility-2 (jdf2) locus, also called runty-jerky-sterile (rjs), was originally identified through complementation studies of radiation-induced p-locus mutations. Studies with a series of ethylnitrosourea (ENU)-induced jdf2 alleles later indicated that the pleiotropic effects of these mutations were probably caused by disruption of a single gene. Recent work has demonstrated that the jdf2 phenotype is associated with deletions and point mutations in Herc2, a gene encoding an exceptionally large guanine nucleotide exchange factor protein thought to play a role in vesicular trafficking. Here we describe the molecular characterization of a collection of radiation- and chemically induced jdf2/Herc2 alleles. Ten of the 13 radiation-induced jdf2 alleles we studied are deletions that remove specific portions of the Herc2 coding sequence; DNA rearrangements were also detected in two additional mutations. Our studies also revealed that Herc2 transcripts are rearranged, not expressed, or are present in significantly altered quantities in animals carrying most of the jdf2 mutations we analyzed, including six independent ENU-induced alleles. These data provide new molecular clues regarding the wide range of jdf2 and p phenotypes that are expressed by this collection of recently generated and classical p-region mutations.

Strains of Salmonella typhimurium that contain the aroC321 allele require phenylalanine, tyrosine, and tryptophan for growth but revert to tryptophan-prototrophy at high frequencies (about 10(-4) per cell plated). The Trp+ derivatives remain auxotrophic for phenylalanine and tyrosine and are genetically unstable, in that they readily give rise to cells that require all three aromatic amino acids. On the basis of growth characteristics and genetic instability, it has been proposed that reversion to tryptophan-prototrophy in aroC321 strains occurs by genetic duplication. This paper provides genetic evidence in support of that hypothesis. The data indicate, moreover, that the tryptophan prototrophs contain a duplication that extends at least from glpT to xyl, a region of greater than 30% of the Salmonella chromosome. The aroC locus is found within the duplicated region, and aroC321/aroC321 merodiploids apparently grow as tryptophan prototrophs because of a gene-dosage effect.